The human genome possesses genes which have the potential, when mutated or expressed at higher than normal levels, to lead to the formation of cancer. The first genes to be identified which had this property were viral oncogenes. The products of these viral oncogenes are responsible for the virus' ability to form tumorigenic cells having altered growth properties. The corresponding cellular oncogenes ("proto-oncogenes") can be activated by viruses, chromosomal rearrangement, gene amplification or mutation.
In all, more than 25 distinct proto-oncogenes have been identified in mammalian cells. See Hunter, Cell, 64: 249-270 (1991). In their normal context, proto-oncogenes have important functions in control of cell growth; they encode growth factors or their receptors (e.g., c-erbB proto-oncogene), they act as intracellular signal transducers (e.g., c-src, c-ras proto-oncogenes), and they are transcription factors that control expression of cellular genes required for cell proliferation (e.g.,c-myc, c-fos, c-jun proto-oncogenes). Proto-oncogene expression is tightly regulated with regard to both the level of expression and the timing of expression during development and during the cell cycle. Activation of proto-oncogenes often occurs through deregulation of their expression.
The genome also expresses genes which block cancer formation. These "anti-oncogenes", also called tumor suppressors, are negative regulators of cell division. The mutation and/or loss of these genes can lead to deregulated cell proliferation and a dramatically increased incidence of cancers. In fact, the most commonly mutated genes in human cancers are tumor suppressors. Individuals heterozygous for germ-line mutations in tumor suppressors are strongly predisposed to one or more types of cancer. The loss or mutation of the normal tumor suppressor gene in these heterozygous individuals can lead to tumor formation.
The first tumor suppressor discovered was the retinoblastoma susceptibility gene (RB) which was identified from studies on sporadic and inherited retinoblastoma. All normal human cells carry one copy of the RB gene on each chromosome 13. Rarely, a mutational event destroys one of the pair, but the cells are still phenotypically normal (i.e., the mutation is "recessive") as the remaining normal RB gene acts to inhibit cellular proliferation and tumorigenic potential. If a mutation or loss of the remaining normal RB gene occurs in these heterozygous cells, a tumor will develop. Tumorigenesis requiring these two mutations is exceedingly rare and only single tumors will develop. Since one of the RB genes is already nonfunctional at birth in the inherited form, single mutations can result in an RB-/- genotype and the likelihood of formation of tumors is far greater.
Mutations in the RB gene have been associated definitively with the occurrence of retinoblastomas. RB deletion or mutation has also been observed in a variety of other human tumors. Most notable among these other cancers are osteosarcoma as well as bone and soft-tissue sarcomas. RB loss or mutation is also strongly implicated in small cell lung carcinoma and, to a lesser extent, other lung cancers and esophageal carcinoma. Functional loss of RB has also been associated with cancer of the bladder, prostate, breast and liver, as well as lymphomas and leukemias.
The RB tumor suppressor protein forms complexes with a variety of cellular proteins that play a role in transcriptional regulation, in particular, the E2F family of cellular transcription factors. See Kovesdi et al., Cell, 45: 219-228, 1986. It is now known that the RB protein associates with E2F, specifically in mid-late G1 and S phase during the cell cycle. E2F-binding sites have been known to exist in a number of growth-regulatory genes. In particular, a subset of these genes encodes products that play a role in DNA synthesis. RB appears to form a complex with E2F which represses transcription of E2F-responsive promoters, thus inhibiting growth. Promoters such as those directing expression of dihydrofolate reductase (DHFR), DNA polymerase alpha, cdc2, and thymidine kinase all contain E2F-binding sites. See, for instance, Means et al., Mol. Cell. Biol., 12:1054-1063 (1992)and Pearson et al., Mol. Cell. Biol., 11:2081-2095 (1991). Inactivation of RB genes may effectively eliminate transcriptional repression of important genes involved in DNA synthesis.
Moreover, at least one E2F member, E2F1, has been demonstrated to be a target of RB action and exhibits oncogenic properties when overexpressed in immortalized cell lines or, in conjunction with activated ras oncogene. See, for example, Singh et al., U J., 13: 3329-3338, (1994) and Johnson et al., Proc. Nat. Acad. Sci. USA 91: 12823-12827 (1994). The possibility exists that, in the case of certain RB-/- tumors, inactivation of RB has led to deregulated E2F activity and/or increased level of E2F expression.
It is theoretically possible to treat human RB (-/-) cancers through re-introduction of a wild-type RB gene. The exogenously delivered tumor suppressor is likely to inhibit tumor proliferation. It is known that re-introduction of the RB tumor suppressor gene into RB-defective tumor cells inhibits the tumor cell growth and inhibits the neoplastic phenotype of the target cells. See, for example, Huang et al., Science, 242: 1563-1566 (1988) and Bookstein et al., Science, 247: 712-715 (1990).
Nevertheless, gene therapy using RB tumor suppressor genes is problematic. Since RB (-/-) tumor cells have already mutated the resident RB gene, it is possible that these cells lacking tumor suppressor gene function have already evolved a mechanism to mutate or destabilize wild-type RB genes and might certainly possess the mechanism to mutate or destabilize the introduced wild-type RB gene.
In particular, it has been shown that several cell lines from tumors that have had the RB gene re-introduced have become very tumorigenic and have formed large, progressively growing tumors when injected into mice. See Zhou et al., Proc. Am. Assoc. Cancer Res., 34: 3214 (1993). This phenomenon is called tumor suppressor gene resistance and might be due to the fact that the tumor cells may have inherited or acquired the ability to mutate or destabilize wild-type RB. Alternately, or in addition, the tumor cells may be able to convert RB proteins to an inactive (i.e., phosphorylated) form that allows tumor growth to continue.
Overexpression of tumor suppressor RB genes is likely to be cytotoxic to both tumor cells and proliferating normal cells. Thus, this therapy may be no more effective than conventional chemotherapy, which indiscriminately kills normal and abnormal cells. Moreover, inhibition of tumorigenicity using this approach is often incomplete and a significant percentage of the RB-reconstituted tumor cells (retaining normal RB expression) still form malignant and invasive tumors in nude mouse tumorigenicity assays. See Xu et al., Cancer Res., 51: 4481-4485 (1991); Banerjee et al., Cancer Res., 52: 6297-6304 (1992).
Accordingly, there is a need in the art for a genetic therapy for tumor or cancer cells which can safely overcome these problems.